- Comparative investigation of the cleavage step in the synthesis of model peptide resins: Implications for Nα-9- fluorenylmethyloxycarbonyl-solid phase peptide synthesis
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Based on our studies of the stability of model peptide-resin linkage in acid media, we previously proposed a rule for resin selection and a final cleavage protocol applicable to the Nα-tert-butyloxycarbonyl (Boc)-peptide synthesis strategy. We found that incorrect choices resulted in decreases in the final synthesis yield, which is highly dependent on the peptide sequence, of as high as 30%. The present paper continues along this line of research but examines the Nα-9-fluorenylmethyloxycarbonyl (Fmoc)-synthesis strategy. The vasoactive peptide angiotensin II (AII, DRVYIHPF) and its [Gly 8]-AII analogue were selected as model peptide resins. Variations in parameters such as the type of spacer group (linker) between the peptide backbone and the resin, as well as in the final acid cleavage protocol, were evaluated. The same methodology employed for the Boc strategy was used in order to establish rules for selection of the most appropriate linker-resin conjugate or of the peptide cleavage method, depending on the sequence to be assembled. The results obtained after treatment with four cleavage solutions and with four types of linker groups indicate that, irrespective of the circumstance, it is not possible to achieve complete removal of the peptide chains from the resin. Moreover, the Phe-attaching peptide at the C-terminal yielded far less cleavage (50-60%) than that observed with the Gly-bearing sequences at the same position (70-90%). Lastly, the fastest cleavage occurred with reagent K acid treatment and when the peptide was attached to the Wang resin.
- Jubilut, Guita Nicolaewsky,Cilli, Eduardo Maffud,Crusca Jr., Edson,Silva, Elias Horacio,Okada, Yoshio,Nakaie, Clovis Ryuichi
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- Resin selection based on the lability of peptidyl-resin linkage towards HF and TFA steps: Dependence on the C-terminal amino acid and peptide length
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Ideally, the solid support used for tert-butyloxycarbonyl (Boc)-peptide synthesis method must allow sufficient stability of the peptide linkage towards TFA-α-amino deprotection but adequate lability to final HF cleavage. Due to these conflicting characteristics, the choice of the correct resin for peptide synthesis is complex and dependent upon many factors. Aiming to clarify this issue, a time-course study of the trifluoroacetic acid (TFA) and HF steps using model peptidyl-resins was developed. The peptidyl-resin bond stability was strongly dependent upon the resin and the carboxy-terminus residue. The decreasing order of acid stability for resins was: benzhydrylamine-resin (BHAR)>p-methylbenzhydrylamine-resin (MBHAR)?4- (oxymethyl)-phenylacetamidomethyl-resin (PAMR)>chloromethyl-resin (CMR) and Phe>Gly?His?Asp for C-terminal amino acids. HF-cleavage times of near 6 h (BHAR) and 23 h (MBHAR and PAMR) were necessary for quantitative cleavage of hydrophobic Phe residue-containing sequence at its C-terminal portion. When premature chain loss in TFA and incomplete cleavage in HF values were both quantitatively considered, a significant decrease in the overall yield (up to 35%) was observed in some resins. Moreover, MBHAR was more suitable than BHAR only when the peptide C-terminal residue is hydrophobic. The data also allow the prediction that due to more significant chain loss in TFA when MBHAR is used, BHAR will be the resin of choice for much longer than 40-mer peptide sequences containing C-terminal hydrophilic residues. Otherwise PAMR is the best resin for the synthesis of free carboxyl peptides but significantly low HF cleavage was observed when the C-terminal amino acid is of the hydrophobic-type.
- Jubilut, Guita N.,Miranda, Maria Teresa,Tominaga, Mineko,Okada, Yoshio,Miranda, Antonio,Nakaie, Clovis R.
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- Ultrafast extraction of proteins from tissues using desorption by impulsive vibrational excitation
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A picosecond IR laser (PIRL) can be used to blast proteins out of tissues through desorption by impulsive excitation (DIVE) of intramolecular vibrational states of water molecules in the cell in less than a millisecond. With PIRL-DIVE proteins covering a range of a few kDa up to several MDa are extracted in high quantities compared to conventional approaches. The chemical composition of extracted proteins remains unaltered and even enzymatic activities are maintained.
- Kwiatkowski, Marcel,Wurlitzer, Marcus,Omidi, Maryam,Nimer, Refat,Horst, Andrea,Schlüter, Hartmut,Ren, Ling,Kruber, Sebastian,Robertson, Wesley D.,Miller, R. J. Dwayne
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- Propargyl-Substituted Thiocarbamoylbenzamidines of Technetium and Rhenium: Steps towards Bioconjugation with Use of Click Chemistry
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A new propargyl-substituted thiocarbamoylbenzamidine has been synthesized. The compound acts as a tetradentate ligand and forms stable complexes with {ReVO}3+and {TcVO}3+cores. Click couplings of the resulting complexes with benzylazide lead to prototype triazole derivatives, which were analyzed by NMR and IR spectroscopy and mass spectrometry, as well as by X-ray diffraction. A similar coupling procedure has been applied to an azido-modified angiotensin-II peptide, which gives the desired rhenium(V) bioconjugate in good yield. The ease of this coupling and the stability of the conjugate recommend such tetradentate thiocarbamoylbenzamidines also for clinically relevant bioconjugates with99mTc.
- Castillo Gomez, Juan Daniel,Hagenbach, Adelheid,Abram, Ulrich
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p. 5427 - 5434
(2016/12/16)
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- Solid-phase peptide synthesis in highly loaded conditions
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The use of very highly substituted resins has been avoided for peptide synthesis due to the aggravation of chain-chain interactions within beads. To better evaluate this problem, a combined solvation-peptide synthesis approach was herein developed taking as models, several peptide-resins and with peptide contents values increasing up to near 85%. Influence of peptide sequence and loading to solvation characteristics of these compounds was observed. Moreover, chain-chain distance and chain concentration within the bead were also calculated in different loaded conditions. Of note, a severe shrinking of beads occurred during the α-amine deprotonation step only when in heavily loaded resins, thus suggesting the need for the modification of the solvent system at this step. Finally, the yields of different syntheses in low and heavily loaded conditions were comparable, thus indicating the feasibility of applying this latter "prohibitive" chemical synthesis protocol. We thought these results might be basically credited to the possibility, without the need of increasing molar excess of reactants, of carrying out the coupling reaction in higher concentration of reactants - near three to seven folds - favored by the use of smaller amount of resin. Additionally, the alteration in the solvent system at the α-amine deprotonation step might be also improving the peptide synthesis when in heavily loaded experimental protocol.
- Nakaie, Clovis R.,Oliveira, Eliandre,Vicente, Eduardo F.,Jubilut, Guita N.,Souza, Sinval E.G.,Marchetto, Reinaldo,Cilli, Eduardo M.
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experimental part
p. 101 - 109
(2011/06/25)
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- The molecular basis for the selection of captopril cis and trans conformations by angiotensin I converting enzyme
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Enzyme-inhibitor recognition is considered one of the most fundamental aspects in the area of drug discovery. However, the molecular mechanism of this recognition process (induced fit or prebinding and adaptive selection among multiple conformers) in several cases remains unexplored. In order to shed light toward this step of the recognition process in the case of human angiotensin I converting enzyme (hACE) and its inhibitor captopril, we have established a novel combinatorial approach exploiting solution NMR, flexible docking calculations, mutagenesis, and enzymatic studies. We provide evidence that an equimolar ratio of the cis and trans states of captopril exists in solution and that the enzyme selects only the trans state of the inhibitor that presents architectural and stereoelectronic complementarity with its substrate binding groove.
- Tzakos, Andreas G.,Naqvi, Nawazish,Comporozos, Konstantinos,Pierattelli, Roberta,Theodorou, Vassiliki,Husain, Ahsan,Gerothanassis, Ioannis P.
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p. 5084 - 5087
(2007/10/03)
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